Abnormalities in the plasminogen activator system have been implicated in the pathogenesis of atherothrombotic disorders. In particular, elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA), and tPA/PAI-1 complexes have been found to correlate with increased risk of myocardial infarction (Ml) and/or stroke. Vascular fibrinolytic balance is, to a large part, determined by the competing effects of t-PA and PAI-1, and reflects a complex interplay between genetic and environmental factors. For the last several years, this laboratory has been involved in a series of basic and clinical investigations that have served to define hormonal and metabolic factors that contribute to the molecular regulation of PAI-1 and its role in the arterial thrombosis and arteriosclerosis. Based on observations made in genetically modified mice, it was hypothesized that the endogenous plasminogen activators, t-PA and u-PA, in conjunction with thrombomodulin, serve as the critical endogenous defenders against thrombosis in the coronary circulation. Accordingly, an excess of PAI-1 would be expected to increase the risk of coronary thrombosis, since PAI-1 inhibits all three components of this defensive triad, i.e. t-PA, u-PA, and APC. Indeed, there is substantial experimental and epidemiological evidence that PAI-1 may in fact contribute to the development of ischemic cardiovascular disease. In studies supported by this R01, we have engineered novel lines of transgenic mice that overexpress a stable form of human PAI-1 and reported that these animals develop age-dependent spontaneous macrovascular coronary thrombosis and subendocardial myocardial infarction. The central hypothesis of this proposal is that vascular PAI-1 excess promotes the development of intravascular thrombosis and atherosclerosis. This application is composed of four specific aims designed to advance these investigations and specifically to clarify the prothrombotic mechanism of PAI-1 by characterizing new transgenic lines that express specific structural mutations in critical functional domains of the PAI-1 protein. Additionally, we intend to investigate the impact of hyperlipidemia on PAI-1-induced coronary thrombosis and to investigate the role of recently developed PAI-1 antagonists in experimental models of arterial and venous thrombosis. PAI-1 is the major physiologic inhibitor of t-PA in plasma, and is elevated in a variety of clinical situations that are associated with increased risk of ischemic cardiovascular events. Recent insights into the biology of PAI-1 suggest that it is more than just an innocent bystander in the pathogenesis of ischemic heart disease. Elevated PAI-1 levels appear to increase the risk of atherothrombotic events and may also promote the progression of vascular disease. It is anticipated that these studies will provide new insights into the mechanisms through which PAI-1 contributes to the development of intravascular thrombosis and vascular pathology and identify new approaches for the treatment of PAI-1-associated vascular disease. [unreadable] [unreadable]